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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
ACS Nano. Author manuscript; available in PMC 2013 November 27.
Published in final edited form as:
PMCID: PMC3508364
NIHMSID: NIHMS415933

Chemical Transformations of Nanosilver in Biological Environments

Abstract

The widespread use of silver nanoparticles (Ag-NPs) in consumer and medical products provides strong motivation for a careful assessment of their environmental and human health risks. Recent studies have shown that Ag-NPs released to the natural environment undergo profound chemical transformations that can affect silver bioavailability, toxicity, and risk. Less is known about Ag-NP chemical transformations in biological systems, though the medical literature clearly reports that chronic silver ingestion produces argyrial deposits consisting of silver-, sulfur-, and selenium-containing particulate phases. Here we show that Ag-NPs undergo a rich set of biochemical transformations, including accelerated oxidative dissolution in gastric acid, thiol binding and exchange, photoreduction of thiol- or protein-bound silver to secondary zero-valent Ag-NPs, and rapid reactions between silver surfaces and reduced selenium species. Selenide is also observed to rapidly exchange with sulfide in preformed Ag2S solid phases. The combined results allow us to propose a conceptual model for Ag-NP transformation pathways in the human body. In this model, argyrial silver deposits are not translocated engineered Ag-NPs, but rather secondary particles formed by partial dissolution in the GI tract followed by ion uptake, systemic circulation as organo-Ag complexes and immobilization as zero-valent Ag-NPs by photoreduction in light affected skin regions. The secondary Ag-NPs then undergo detoxifying transformations into sulfides, and further into selenides or Se/S mixed phases through exchange reactions. The formation of secondary particles in biological environments implies that Ag-NPs are not only a product of industrial nanotechnology, but have long been present in the human body following exposure to more traditional chemical forms of silver.

Keywords: silver, selenium, nanomaterial transformation, fate, health risks, photochemistry

Engineered nanomaterials can undergo profound transformation between the time of synthesis and the time at which human or environmental receptors are exposed.14 These material transformations may involve adsorption, reaction, dissolution, or aggregation, and can affect transport, bioavailability, bioaccumulation, toxicity, and ultimately risk.

A number of recent studies have focused on the environmental transformations of silver nanoparticles (Ag-NPs).520,23 These studies show that Ag-NPs undergo slow oxidative dissolution by molecular oxygen and protons,5,18,19 reactions with reduced sulfur species or chloride,7, 8, 1012, 17 adsorption of polymers,20 natural organic matter (NOM),15,16,20 or proteins,21,22 and aggregation that depends on media and coatings.14,15,17,20,23 The transformed products can show reduced biological activity (e.g. silver sulfide)24,25 or enhanced biological activity (e.g. the free ion or soluble complexes)26,27 relative to the initial particle. In the case of silver-based nanomaterials, it has become quite clear that chemical transformations must be considered in any realistic risk assessment.28,29

Less is known about Ag-NP transformations in biological systems. The chemical environments in biological systems have some features in common with those in the natural environment (presence of Cl, some organic ligands, reduced sulfur species), but also significant differences. Some biological environments have very low pH (gastric fluid), very high concentrations of organic ligands including thiols, significant concentrations of selenium in addition to sulfur, and the potential for photochemistry in the near-skin region. Biological compartments also contain ion channels and pumps, which can transport silver ion by mechanisms evolved in nature for transport of sodium30 or copper ion.31 Many aspects of Ag-NP biochemistry are unexplored, such as the interactions with selenium species, the photochemistry of Ag-biocomplexes in the near-skin region, or are incompletely understood, such as the extent of dissolution in the GI tract.32

The medical literature does not systematically address the chemical mechanisms and pathways of Ag-NP transformation, but it does provide useful information on the final fate and form of silver in the human body. Overexposure to silver through ingestion, inhalation, and dermal contact can raise silver concentrations in blood, and in some cases cause irreversible skin discoloration (argyria) especially in sun-exposed areas.3346 Silver rich granules, mostly collocated with sulfur and selenium, have been detected in the connective tissue of the dermis in argyria patients.33,34,36,41,42,4446 A recent study reported the deposition of Ag-, S- and Se-containing nanoparticles in small intestine, liver and kidney tissue after oral exposure of rats to Ag-NPs.47 It is clear that a significant long-term fate of silver in the body is as particulate deposits, and the deposition is favored by light-exposure, but the chemical pathways have not been systematically investigated and remain unclear.

Here we study the chemical transformations of Ag-NPs in biological media with emphasis on compartments in the human body. We report accelerated dissolution in gastric acid, photoreduction of Ag-thiol and Ag-protein complexes to metallic Ag-NPs, and the ability of reduced selenium species to react with Ag surfaces and to exchange with sulfur in silver surfaces exposed to prior sulfidation. The combined results suggest a pathway for argyrial deposits that involves partial gastric digestion to soluble silver, ion uptake and systematic transport as thiol complexes, photoreduction of Ag(I) to immobilize silver in the form of Ag-NPs in the near-skin region, and then in situ transformation to sulfides and selenides. We thus propose that argyrial deposits are secondary particles rather than translocated primary particles, and as such, are not unique to Ag-NP exposure, but occur upon exposure to a variety of silver compounds and silver containing materials.

RESULTS

Because of the wide range of nanosilver applications, a variety of exposure routes are relevant including ingestion, inhalation, dermal contact, wound surface application, and insertion or implantation of medical devices.48 These exposure routes bring Ag-NPs in contact with a range of different fluid environments, and we begin by examining oxidative dissolution at the initial point of entry.

Oxidative Dissolution in Biological Media

Nanosilver is unstable to oxidation and releases ions through gradual reaction with dioxygen and protons or dissolution of preexisting oxide films in fluid media. Oxidative dissolution is a complex chemical reaction influenced by pH, coatings, and ligands in the surrounding fluid,5,6,8,11,12,14,16,18,26,49 and has been extensively characterized in environmental fluid phases,5,11,12,14,16,49 but less so in biological media,6,11,50 though the important role of released silver ions in Ag-NP toxicity is well recognized.26,27,51,52 Biological compartments cover a wide pH range (1 – 8), and we expect pH5,19 and particle surface area (particle size and aggregation state)6,18,19,27,52,53 to be important factors influencing biodissolution rates. Figure 1 explores the effects of pH and primary particle size in four media representing the: (1) stomach (pH 1.5),54 (2) lysosome (pH 4.5),55 (3) inflammatory phase of acute wounds (pH 5.7),56 and (4) extracellular environment/blood/lung fluid (pH 7.4),54 using previously published kinetics for a model citrate-capped Ag-NP material system:5

Figure 1
Effects of pH and particle size on Ag-NP oxidative dissolution in simple media estimated using previously published kinetics.5 (A) pH effect for 5 nm diameter Ag-NPs. (B) Primary particle size effect at pH 1.5. The calculations use the kinetic law of ...

equation M1
Eq. 1

where E = 77 kJ/mol, A = 2.5 × 1013 day−1 and n = 0.18. An area normalized form useful for estimating the size dependence has (1/S)(−dm/dt) on the left-hand side and a preexponential factor A = 7.6 × 1016 μg-release/day-m2-particle-surface. Figure 1 suggests that the low pH in gastric fluid will lead to accelerated dissolution of Ag-NPs in the stomach, but that dissolution will be incomplete for most particles due to limited residence time (10 – 240 min in stomach).57 Ligand, coating, and salt effects limit our ability to accurately predict dissolution rates in complex media at this time from simple approaches such as the use of Eq. (1), so experimental data on gastric fluid and wound fluid simulants are needed to confirm this conclusion. Our previous work used ultrafiltration/atomic-adsorption (AA) for release rate measurement, which is quantitative but incompatible with the presence of proteins or other macromolecules, which can bind Ag+ and be removed by the ultrafilter, leading to incorrect assignment of Ag to the condensed (particle) phase. At high silver doses (above those relevant to the natural environment) AgCl(s) may also form and be removed by ultrafiltration, and chloride has been reported to cause matrix effects that interfere with atomic absorption method.58 For experiments in biological media, therefore, we need an alternative technique, and chose to adapt an in situ plasmon resonance tracking method.11,59

Ag-NPs are well known to exhibit strong localized surface plasmon resonance (LSPR), and the corresponding UV-vis absorption peak is sensitive to particle size, shape, aggregation state, and the external dielectric environment,60 and can be used to monitor total Ag concentration.59,61 We adapted the technique of Espinoza59 and Zook11 with modification by using 1 wt% polyvinylpyrrolidone (PVP) to protect particles from aggregation and UV-vis absorbance to track ion release. The technique was first validated by oxidatively dissolving Ag-NPs in 0.084 M HNO3, which gives the same pH as synthetic gastric acid. Figure 2A shows a rapid decrease of the LSPR signal at ~ 397 nm, corresponding to Ag0 oxidation. Assuming optical absorption at the LSPR peak height is proportional to total Ag0 concentration59 gives a release curve that can be compared to direct measurement by ultrafiltration/AA (Fig. 2B). Figure 2B validates the LSPR technique, which can now be used in complex media containing Ag-binding macromolecules that cannot pass the ultrafilter (MW cutoff 3K).

Figure 2
Development and validation of plasmon resonance tracking technique for Ag-NP (average diameter 5 nm) dissolution in complex media. (A) UV-vis spectra recorded during incubation of Ag-NP in dilute HNO3, showing rapid decrease of peak absorbance due to ...

Figure 3 uses the LSPR technique to measure silver ion release profiles in gastric fluid and pseudo-extracellular fluid (PECF) with BSA as a simulant for wound exudate. Dissolution is relatively rapid in gastric fluid in agreement with the behavior in simple media (dashed black curve) and is very slow in PECF. The elevated pH in PECF is not sufficient to explain the slow dissolution (see dashed red curve), and may be related to ligands in the PECF buffer. Interestingly, the presence of albumin greatly accelerates ion release. It is possible that the strong affinity of BSA for Ag+ 62 facilitates the mobilization of surface bound Ag+ to the solution phase though more work is needed to understand this mechanism.

Figure 3
Time-resolved silver ion release from Ag-NPs (average diameter 5 nm) in synthetic gastric fluid (pH 1.12) and wound fluid (pH 7.52). The black and red dashed lines represent the calculated silver ion release in simple media pH 1.5 and 7.4 buffer, respectively. ...

Interaction of Ag+ with Chloride and Thiol

The Ag+ produced by oxidative dissolution can further transform in biological media, whose composition determines the speciation, mobility and bioactivity of silver. The free Ag+ concentration in biological media is extremely low due to complexation and possibly precipitation of Ag+ with Cl, typically on the order of 10−9 M (Ksp=1.77×10−10 for AgCl).65 The maximum concentration of soluble silver species (sum of Ag+, AgCl(aq), AgCl2 and AgCl32−) estimated using visual MINTEQ (version 3.0)66 is 0.51 mg/L and 0.58 mg/L in synthetic gastric acid and albumin-free wound simulant, respectively. Above this concentration, AgCl(s) will appear and limit further increases in silver bioavailability.67 In many exposure scenarios the total silver dose is less than these values (< 0.5 ppm), so AgCl(s) is not expected as a product, but it can occur at high doses and especially in toxicity studies, where elevated doses are used to see acute effects.68,69

The Ag+ and its soluble complexes in the GI tract can be taken up into systemic circulation by active transport routes for Na+ and Cu+ 70 and enter the bloodstream, where it is expected to bind to proteins and distributed to a variety of tissues and organs.71 Serum albumin, the most abundant plasma protein, is involved in the transport of other ions including Cu2+, Ni2+, Zn2+, Co2+.7275 The affinity of human serum albumin (HSA) towards Ag+ and the abundance of HSA in blood (50 g/L)76 make serum albumin a likely transporter for Ag+. Binding sites for Ag+ on HSA have been reported with the formation constants of 105 and 104, and cysteine, methionine and disulfide bridge residues are the major functional groups involved.77 In general, the soft acid Ag+ binds strongly to thiol (-SH) groups (log K formation = 11.9 for Ag+-cysteine)78 including small molecule thiols such as reduced glutathione (GSH).79 GSH is a primary endogenous antioxidant with a typical blood concentration of 1 mM.80 Here we use GSH as a model thiol compound to investigate Ag+ binding, exchange, and competition with chloride species in biological fluids.

We first observed that free Ag+ and free GSH show no coexistence region in DI water (SI, Fig. S1) consistent with the strong binding reaction: Ag+ + GSH↔GS-Ag + H+.81 At high GSH/Ag ratio the stoichiometry of the complex is Ag-GSH, while at low ratios the stoichiometry approaches Ag2-GSH. The removal of reaction products using centrifugal ultrafiltration (MW cutoff 3K) indicates the formation of high molecular weight silver-GSH polymer complexes. Silver-thiol polymers were reported to have a two-dimensional layered structure with Ag-S bonds in a three-coordinate mode,82 and silver-rich complexes were also identified where excess silver binds to sulfur groups or forms Ag-Ag bonds with mercaptide-bound silver atoms.83,84 More information on the formation range of silver-thiol polymers is found in the Supporting Info.

An important question for biological partitioning is whether Ag+ can exchange easily between high affinity ligands such as Cl and RSH and remain mobile. To test chloride/thiol exchange, we first formed AgCl precipitates and exposed them to GSH while tracking particle size by dynamic light scattering (DLS). Figure 4A shows that addition of AgNO3 to PBS buffer causes rapid precipitation of AgCl, and the particle size increases quickly from ~300 nm to microns. Addition of GSH (Fig. 4B) dissolves the AgCl precipitates within 45 min to produce Ag-GSH complexes that can be monomers or soluble oligomers undetectable by DLS. We note that silver-GSH complexes have been shown to be bioavailable to aquatic organism,85 and were reported to enhance silver transport from AgCl across simulated biological membranes.86 Silver is also reported to readily exchange between different thiol groups87 despite the strong Ag-thiol bond. Well-dispersed silver-GSH complexes are likely important transporters that deliver silver to biological targets through thiolate ligand exchange.87 By this mechanism, silver can be transported and distributed across multiple tissue and organ systems following systematic absorption.71 Of special interest here are clinical cases of argyria where silver is located in the basement membrane of skin as particles,34, 36, 38, 41, 42, 44 with unknown formation mechanism.

Figure 4
Demonstration of Ag+ interactions with chloride and preferred binding to thiol. (A) Time-resolved measurement of hydrodynamic particle sizes during formation and growth of AgCl particles upon addition of AgNO3 into PBS buffer. (B) Further time-resolved ...

Biological Photochemistry of Silver

Argyria, characterized by the irreversible bluish-gray discoloration of skin, is most prominent in light affected skin areas, and has been characterized as a particulate phase with Ag collocated with S and/or Se.34,36,38,41,42,44,45 The clinical pattern of sunlight dependence suggests the importance of a photocatalyzed reaction. Here we start with the silver-thiol and silver-protein complexes described in the previous section, and study their behavior under UV-visible light.

Figure 5A shows that clear silver-GSH complexes in DI water gradually darken over 6 h under 365 nm UV irradiation but not in room light. Figure 5B shows full spectra, which develop a broad 400–550 nm band over time under UV irradiation but not in room light. TEM (Fig. 5C and Fig. S2) and XRD (Fig. 5D) show this process is accompanied by nanoparticle formation in FCC Ag0 phase. Similar photo-transformations were also observed in PBS buffer (Fig. 6), and in selected experiments using different GSH/Ag+ ratios or using cysteine and oxidized glutathione (GSSG) as thiol sources. In each case the product was zero-valent nanosilver (SI, Figure S3). Note that prolonged incubation of AgNO3/GSH for several days in room light or dark causes a similar color change to that in Fig. 5, which must be uncatalyzed GSH reduction88 that may occur in deep dark tissue, but more slowly.

Figure 5
UV irradiation induces Ag-NP formation in AgNO3 / GSH mixtures at concentration ratio of 1/0.3 in DI water. (A) Photographs after 1-6 h light exposure show nanoparticle formation only at UV wavelengths. (B) UV-vis spectra of samples in (A). (C) TEM image ...
Figure 6
UV irradiation induces formation of small-diameter Ag-NPs from AgNO3 / GSH mixtures at a concentration ratio of 1/1 in PBS buffer. (A) Photograph of the mixture after exposed to UV for 3 h and 12 h. (B) TEM image of 12 h UV exposed sample in (A). Experiments ...

It is clear that UV irradiation of Ag complexes with small molecule thiols produces Ag-NPs. Much of the thiol content in blood and tissue is found in protein, and the main component of basement membrane connective tissue, where argyrial deposits are often found, is the protein collagen. Here we exposed Type I collagen solutions to AgNO3 and UV and observed irregularly shaped silver nanostrucutres (Fig. 7A and 7B). SAED pattern identifies the phase again as polycrystalline FCC Ag0 (Fig. 7B). Figure 7C gives UV-vis spectra, showing that photoreduction progresses over time and even occurs to some extent under room light. Photoreduction of AgNO3 in collagen solution is fast - the fraction of input Ag+ that is reduced increases from 30% to 50% between 1 h and 5 h exposures (Fig. 7D). Experiments using a gel form of collagen that more closely mimics the physical form of tissue also provide evidence of Ag-NP formation under UV-vis light with no transformation in the dark (SI, Fig. S4).

Figure 7
Photoreduction produces Ag-NPs in collagen solution. (A) Photograph of AgNO3 (1 mM) in collagen solution (0.1 mg/mL) at pH 3.9 and 7.4 after UV irradiation for 3 h. (B) TEM image of 3 h UV exposed sample at pH 7.4, the inset gives SAED pattern corresponding ...

Together, these data show that a range of thiol-containing biomolecules and proteins produce zero-valent Ag-NPs upon UV photodecomposition with slower rates in visible light. The chemistry demonstrated here is related to that in black-and-white photography and in biological silver staining.89 Because these decomposition products are zero-valent Ag rather than sulfide phases, the photodecomposition alone is not a sufficient explanation for the known composition of argyrial deposits and an additional mechanism is needed (next section).

Sulfide and Selenide Reactions

We thought originally that Ag/S/Se argyrial particles may come directly from Ag-thiol photodecomposition, but the data show that the photodecomposition products are not sulfides but zero-valent Ag particles. It has been established that Ag2S-NPs can form from direct sulfidation of such Ag-NPs under conditions relevant to the natural environment.710 Sulfides are present in the human body at concentrations reported from the nanomolar range90 up to 10 – 100 μM,91 and the corresponding sulfidation time scales range from hours to days,7 making Ag-NP sulfidation a plausible transformation in biological systems as it is in the natural environment. Because previous literature has focused on Ag-NP sulfidation reactions, we focus here on Ag-NP reactions with selenium, which are unique to the biological setting.

The pathways leading to co-localization of Ag and Se in argyrial deposits were initially unclear. We found no literature on Ag/Se biochemistry, through selenium reactions with silver nanoclusters are the basis for the “selenium toning” technique used for stylistic and stability enhancement in black-and-white photography.92 We designed experiments to see if selenium can “tarnish” silver surfaces in a manner similar to sulfur.7 Because selenide oxidizes more readily than sulfide, we used selenite (SeO32−) and NaBH4 to generate reduced selenium (Sex−, x=1 or 2) in situ93 in anaerobic atmospheres in a glove box (with continuous measurement of O2 at < 0.2 ppm). Addition of silver foil to this solution leads to visible tarnish within 30 min, and the XRD spectrum after one week shows the formation of crystalline silver selenide (Figure 8). The route to Ag2Se may run through Se2− and trace O2 in analogy to oxysulfidation.7 More likely at this low oxygen content is partial reduction of selenite by sodium borohydride to diselenide94 followed by disproportionation to Ag2Se and Se0. We do observe the characteristic red nanoparticles of Se0 as a byproduct in some experiments. Both selenide and diselenide are important intermediates in human selenium metabolism.95

Figure 8
“Tarnishing” of metallic silver foil with reduced selenium species generated in situ in aqueous media. (A) Optical image of silver foil (99.9% Ag) incubated with DI water (right) and reduced selenium (left) for 7 days, showing black scale ...

The much lower abundance of Se in the human body relative to S (~1/10,000)96, 97 makes it unlikely that selenium could compete kinetically with sulfur in its reaction with silver surfaces. This kinetic advantage suggests initial formation of silver sulfides, but this would require another, separate mechanism to explain the incorporation of Se in argyrial deposits. Silver sulfide is highly insoluble (Ksp = 5.92×10−51 for Ag2S),28 but silver selenide even more so (Ksp = 3.1×10−65 for Ag2Se),98 which makes the replacement reaction thermodynamically favorable:

equation M2
Eq. 2

One might expect that the very low solubility of Ag2S would make this reaction slow unless it proceeds through a solid-state mechanism. We were unable to find data on the extent or mechanisms of Se incorporation into Ag/S phases to evaluate the possibility of this exchange reaction. We therefore conducted experiments, in which Ag2S surfaces were created and exposed to selenide generated in situ. The Se/S exchange reaction with sulfidated silver foils was readily observed: within one day selenium replaces sulfur on S-tarnished silver films as determined by EDS (Table S1, SI). The exchange reaction was studied in detail for Ag2S nanoparticles by pre-sulfidating Ag-NPs and incubating them in reduced selenium solutions at equal Se/S molar ratios. The XRD spectra in Fig. 9 show that the Se/S exchange reaction is complete after 3 days and the final product is Ag2Se with no detectable Ag2S. The preference of Ag(I) for Se over S is both thermodynamically predicted and observed in experiment, and the exchange reaction goes to completion over times easily accessible in laboratory studies.

Figure 9
Demonstration of Se/S exchange reactions in Ag-containing nanoparticles. (A) XRD pattern of pristine silver nanopowder (20 – 40 nm, QuantumSphere), giving FCC metallic silver. (B) XRD spectrum of sulfidation product, showing conversion to acanthite ...

SUMMARY AND CONCLUSIONS

The goal of this study was to investigate the chemical pathways for Ag-NP transformation in biological media relevant to human exposures. Our results show accelerated oxidative dissolution in the GI tract, preferential thiol binding and exchange reactions, photodecomposition of Ag-biocomplexes to zero-valent Ag-NPs, and reactions with sulfur and selenium. Particularly interesting findings are the selenium tarnishing of silver surfaces and the ability of selenide to rapidly replace sulfide in Ag2S-NPs and Ag2S films through exchange reaction.

Of particular interest in this study were the chemical pathways leading to argyria. The medical literature provides multiple cases studies that document argyrial deposits as Ag/S/Se particulate phases located in the near-skin region, and that deposition occurs preferentially in light-affected regions. The combined results of the present study allow us to propose a conceptual model for argyrial pathways (Fig. 10). Ingestion or inhalation followed by macrophage clearance can bring Ag-NPs into the GI tract. The very low pH in the stomach lead to ion release but the short residence time should cause the extent of dissolution to be incomplete in many cases. Silver ion and its complexes are brought into the blood stream through ion or nutrient uptake channels and circulate systematically. Note that NPs can also enter body directly through wounds, implants, damaged skin, lung translocation, or through GI tract, but the ability of particles to cross the gut epithelium is limited99 and in this case ion uptake is likely the main route to systemic circulation (vida infra).

Figure 10
Summary of the biological transformations of Ag-NPs and conceptual model for formation of argyrial deposits. The Ag/S/Se argyrial particulate is proposed to be the result of gastric dissolution, ion uptake, circulatory thiol transport, photoreduction ...

The majority of silver in circulation is predicted to be bound to thiol complexes, which have high binding affinities, but are easily exchangable, giving Ag(I) a significant biomolecular mobility. (We note that sulfides and selenides have even higher binding affinities than thiols, but are orders of magnitude lower in concentration in physiological fluids, so binding kinetics strongly favor complexation of silver to thiol as a first step in the pathway). The Ag(I) that reaches the near skin region in light affected areas can be easily photoreduced to metallic Ag-NPs, which effectively immobilizes the silver. The immobilization is both physical, due to low particle diffusivity, and chemically, since the thiol exchange reactions of Ag(I) are not possible with Ag(0). Once formed, the Ag-NPs transform to sulfide phases in analogy to the documented environmental transformations.710 Possibly unique to biological systems is the incorporation of selenium. The low concentrations of selenium relative to sulfur make it difficult for selenium to complete kinetically with sulfur, but over longer times the higher affinity of Se for Ag (lower Ksp, free energy) predict selenides as the equilibrium state. Our results demonstrate for the first time the ability of selenide to exchange with sulfide in silver phases, giving a plausible route for the known formation of biological silver selenides in argyrial deposits. Both the sulfide and selenide transformations can be regarded as biological detoxification reactions due to the low bioavailability of silver in the highly insoluble products.

One implication of these results is that argyrial deposits are primarily secondary particles rather than translocated primary particles. As such, they are not unique to Ag-NP exposure, but occur upon exposure to a variety of silver compounds and silver containing materials. This secondary particle idea and the proposed dissolution-complexation-photoreduction-sulfidation/selenation pathway in Fig. 10 is consistent with the observed fact that some argyrial cases occur in patients who have ingested silver forms that do not appear to contain particles.33,34,100102 Particularly relevant support for our proposed pathway is found in a recent article in this journal,102 in which rats were orally exposed to both soluble silver or two different silver nanoparticles. The biodistribution patterns were observed to be similar for soluble and NP forms, and particles were observed in tissue even when the rats were only fed soluble (non-particulate) silver. The authors conclude that the particles in tissue must be formed in vivo, and that the combined evidence suggests ionic silver is the main bioavailable form following oral ingestion of silver salts or silver NPs.102

The spontaneous formation of silver-containing nanoparticles in this and other studies103105 remind us that Ag-NPs are not only the product of industrial nanotechnology, but may form spontaneously in environmental or biological systems following exposure to other, more traditional chemical forms of silver.

MATERIALS AND METHODS

Materials

Citrate-stabilized Ag-NPs with average diameter of 4–5 nm were synthesized by borohydride reduction.5 Typically, a 59.2 mL solution containing trisodium citrate (0.6 mM) and NaBH4 (2 mM) was prepared in DI water (Millipore, 18.3 MΩ·cm). While vigorously stirring in an ice bath, a 0.8 mL solution of 15 mM AgClO4 was quickly added into the mixture followed by 3 h additional stirring. The resulting brownish yellow Ag-NP suspension was purified with 2 cycle DI water wash using centrifugal ultrafiltration (Amicon Ultra-15 3K, Millipore, MA), concentrated to ~40 mg/L, and stored at 4 °C in the dark for later use. Silver nanopowder (average diameter 20 – 40 nm) manufactured by a vapor condensation process (QuantumSphrere, CA, USA) and silver foil (Strem Chemicals, MA, USA. 99.9% Ag, 0.127 mm thickness) were used as reference silver materials. Type I collagen (10.12 mg/mL in 0.02 M acetic acid) from rat tail tendon was purchased from BD Biosciences (NJ, USA). 10× Phosphate buffered saline (PBS) was obtained from Fisher Scientific (MA, USA) and diluted with DI water to 1× concentration before use. ThioGlo-1, a maleimide reagent that used as fluorescent probe for measuring GSH concentration, was purchased from Calbiochem, Inc. (CA, USA)

Ag-NP Oxidative Dissolution in Simulated Biological Media

Ag-NP oxidative dissolution experiments were conducted in synthetic gastric acid and wound fluids. The chemical compositions of the media are listed in Table 1. Pseudo-extracellular fluid (PECF) and BSA supplemented PECF were used to simulate wound exudates.63,64 A solution containing 10 mg/L Ag-NPs and 2 wt% PVP was first prepared by adding fresh PVP solution (10 wt%) into citrate-stabilized Ag-NP stock suspension, after incubation at 4 °C for 5 min, this solution was mixed with equal volume of 2× concentrated synthetic biological fluids to produce testing solution with Ag-NP concentration of 5 mg/L and PVP concentration of 1 wt%. The Ag-NP suspensions were then incubated at 37 °C in the dark for up to 24 hours, during which period silver ion release was monitored by measuring the localized surface plasmon resonance (LSPR) absorbance using UV-vis spectrometry. Typically, an aliquot of Ag-NP suspension was taken during incubation and the UV-vis spectra was recorded between 300 – 800 nm on a V-630 Spectrophotometer (Jasco, MD) using particle free media as background. The peak shift was negligible (< 5 nm) over the course of the experiment, indicating PVP successfully protects particles from aggregation. The Ag-NP suspension gives LSPR peak at ~ 397 nm, and the absorbance is given by Beer’s law, Alc, where ε is the absorptivity, l is the path length, and c is Ag0 concentration. The mass percentage of Ag-NP dissolution can be derived by

Table 1
Synthetic Biological Fluids
equation M3
Eq. 3

Control experiments using Ag-NP dissolution in HNO3 were performed to validate this method. A test solution containing 5 mg/L Ag-NP, 1 wt% PVP was prepared in 0.084 M HNO3 (the same proton concentration as in synthetic gastric acid). During incubation in the dark at room temperature, silver ion dissolution was quantified both with LSPR absorbance based method as described above or graphite furnace atomic absorption spectrometry (AA) (PerkinElmer AAnalyst 600) after removal of Ag-NPs using centrifugal ultrafiltration (Amicon Ultra-15, 3K).

Silver-Glutathione Complex Formation

The stoichiometry of silver-GSH complex formation was measured by tracking the concentration change of AgNO3 and GSH using AA and ThioGlo-1 fluorescence assay, respectively. A kinetic experiment was first performed by mixing AgNO3 and GSH both at final concentration of 1 mM in DI water, followed with quantification of soluble silver using AA after collection of aqueous phase by centrifugal ultrafiltration. Based on the rapid reaction kinetics, 30 min was used for the dose dependent experiments. Typically, the final AgNO3 concentration was fixed at 1 mM and GSH was added into AgNO3 at concentration of 0.1 to 4 mM, after incubation in the dark at room temperature for 30 min, the aqueous phase was isolated with ultrafiltration filter. An aliquot of the filtrate was analyzed by AA for soluble silver concentration, and another aliquot was used to measure the GSH concentration using ThioGlo-1 fluorescence reagent.107 Details on the fluorescence assay are provided in SI.

The decomposition of AgCl precipitate by GSH was studied in PBS buffer. AgNO3 was first added into PBS buffer at 1 mM, and the immediate formation and growth of AgCl precipitates were monitored using dynamic light scattering (DLS) on a Zetasizer Nano ZS system (Malvern Instruments). After 30 min, GSH was added into the AgCl precipitate at 1 mM, the white precipitate gradually disappeared and the hydrodynamic size of the decomposed particles was tracked with DLS for up to 1 h.

Photodecomposition of Silver Complexes

The further transformation of silver after dissolution and complex formation was investigated by exposure of various test solutions containing 1 mM AgNO3 to UV light. The thiol (-SH) or disulfide (-S-S-) compounds tested included glutathione (0.1 mM, 0.3 mM and 0.5 mM), cysteine (0.3 mM), and oxidized glutathione (0.3 mM). Typically, 5 mL of test solution in a 10 mL glass beaker were prepared by mixing the thiol or disulfide with AgNO3 in DI water, and then subjecting to UV irradiation for 0 – 24 h. Selected experiments with GSH (1 mM) were conducted in PBS buffer, and a control experiment was carried out using 1 mM AgNO3 in DI water. A high intensity UV lamp (B-100AP, UVP LLC, CA) was used to provide 365 nm long-wave UV with an average intensity of ~ 10 mW/cm2. A glass cover was used to prevent water evaporation, and the UV intensity was measured each time before experiment using a UVX Radiometer (UVP LLC, CA) to guarantee the consistency of the irradiation intensity.

AgNP formation was also examined in collagen solution and in a collagen gel matrix. Collagen working solutions were prepared by diluting vendor’s stock with DI water to 0.1 mg/mL, and the resulting solution was acidic with pH of 3.9 (Orion 8165BNWP electrode, Thermo Scientific), then 1 M NaOH was used to adjust pH to 7.4, and AgNO3 was added to the collagen solutions at 1 mM, followed by UV irradiation (365 nm, ~ 10 mW/cm2). Selected experiments were carried out in the dark or ambient lab light. The progress of photodecomposition was monitored by tracking the decrease of soluble Ag+ concentration using AA after removal of particulate phase with centrifugal ultrafiltration. Selected experiments were conducted in collagen gel matrix to better reflect the physical state of tissue. The collagen gel was prepared following vendor’s protocol with modification. Typically, collagen stock solution was diluted with DI water to 4 mg/mL, and AgNO3 was added to concentration of 1 mM, then 1 M NaOH was used to adjust pH to 7.4. The viscous mixture was transferred into 24 well plate and incubation at 37 °C for 1 h, during which period solidified collagen gel was formed. The collagen gel was then incubated in the dark, under the ambient room light, sunlight, or UV lamp for up to 24 h.

Selenium Reactions with Ag0 and Ag2S

Reduced selenium species (Sex−, x=1, 2) were generated in situ by reacting Na2SeO3 with NaBH493 under anaerobic atmosphere in a glove box (O2 ~ 0.1 ppm, Vacuum Atmospheres, CA). Typically, NaBH4 was mixed with Na2SeO3 at 5/1 molar ratio in deoxygenated DI water for 30 min. The formation of Ag2Se on Ag0 surfaces was first investigated using silver foil (99.9% Ag) of 0.127 mm thickness cut into 4 mm × 4 mm pieces. To remove possible oxide layers, the small foil sections were dipped into 1% HNO3 for 5 min before use. A piece of silver foil was incubated in 5 mL of in situ reduced selenium species ([Se] = 5 mM) for 7 days in the glove box. Silver foil incubated in DI water was used as control. Se/S exchange reaction was conducted by treating pre-sulfidated silver nanopowder or silver foil with reduced selenium species. A piece of silver foil or silver nanopowder (10 mM) were first sulfidated with 5 mL of 5 mM Na2S solution for up to 3 days, followed with purification of sulfidation product with DI water wash using centrifugation. The Se/S exchange was achieved by incubating the obtained sulfidated products in reduced selenium ([Se] = 2.5 mM or 5 mM) for up to 3 days, followed by sample purification.

Sample Characterization

The UV-vis spectra of aqueous samples were recorded on a V-630 Spectrophotometer (Jasco, MD) between 300 – 800 nm. The morphology and size of UV induced Ag-NP were determined with transmission electron microscopy (TEM) on a Philips EM420 at 120 kV. TEM samples were prepared by placing a drop of purified sample solution on copper grids with a continuous carbon film coating, followed by solvent evaporation at room temperature overnight. The composition and phase of UV irradiation product were identified by X-ray powder diffraction (XRD) spectrometry on a Bruker AXS D8-Advanced diffractometer with Cu Kα radiation (λ = 1.5418 Å). To prepare the XRD sample, a mixture solution containing 1 mM AgNO3 and 0.3 mM GSH was exposed under UV lamp for 3 h, after sample wash with DI water using centrifugal ultrafiltration, the obtained concentrated nanoparticle suspension was added on a small glass slide and dried overnight in the dark. The Se tarnished silver foil surface and the products of Se/S exchange reaction were analyzed by XRD after sample purification with DI water washing and by energy-dispersive X-ray spectroscopy (EDS) on a LEO 1530 field-emission scanning electron microscope (FE-SEM) at 20 kV accelerating voltage and 8.5mm working distance (see SI). Samples for SEM and EDS were prepared on silicon substrate, and silver sulfide films before/after selenation were placed directly on the conductive tape, both of which meet the requirement of flat surfaces in the ZAF correction method.

Supplementary Material

1_si_001

Acknowledgments

Financial support was provided by NSF grant ECCS-1057547, and the Superfund Research Program of the National Institute of Environmental Health Sciences, P42ES013660. Although sponsored in part by NIEHS, this work does not necessarily reflect the views of the agency.

Footnotes

Supporting Information Available: ThioGlo-1 fluorescence assay used for quantification of GSH concentration, experimental phenomena of silver-GSH polymer formation over a wide range of GSH concentration, reaction stoichiometry of silver-GSH polymer formation, TEM image of Ag-NPs produced by UV irradiation of AgNO3/GSH mixture, Ag-NPs produced by UV irradiation of AgNO3/thiol mixture, photographs of AgNO3 in collagen gel matrix after incubated in the dark, exposure under ambient lab light, sunlight, and UV light, and elemental analysis of sulfidation and Se/S exchange reaction products. This material is available free of charge via the Internet at http://pubs.acs.org.

References

1. Wiesner MR, Lowry GV, Alvarez P, Dionysiou D, Biswas P. Assessing the Risks of Manufactured Nanomaterials. Environ Sci Technol. 2006;40:4336–4345. [PubMed]
2. Lowry GV, Gregory KB, Apte SC, Lead JR. Transformations of Nanomaterials in the Environment. Environ Sci Technol. 2012;46:6893–6899. [PubMed]
3. Lin D, Tian X, Wu F, Xing B. Fate and Transport of Engineered Nanomaterials in the Environment. J Environ Qual. 2010;39:1896–1908. [PubMed]
4. Nowack B, Ranville JF, Diamond S, Gallego-Urrea JA, Metcalfe C, Rose J, Horne N, Koelmans AA, Klaine SJ. Potential Scenarios for Nanomaterial Release and Subsequent Alteration in the Environment. Environ Toxicol Chem. 2012;31:50–59. [PubMed]
5. Liu J, Hurt RH. Ion Release Kinetics and Particle Persistence in Aqueous Nano-Silver Colloids. Environ Sci Technol. 2010;44:2169–2175. [PubMed]
6. Liu J, Sonshine DA, Shervani S, Hurt RH. Controlled Release of Biologically Active Silver from Nanosilver Surfaces. ACS Nano. 2010;4:6903–6913. [PMC free article] [PubMed]
7. Liu J, Pennell KG, Hurt RH. Kinetics and Mechanisms of Nanosilver Oxysulfidation. Environ Sci Technol. 2011;45:7345–7353. [PMC free article] [PubMed]
8. Levard C, Reinsch BC, Michel FM, Oumahi C, Lowry GV, Brown GE. Sulfidation Processes of PVP-Coated Silver Nanoparticles in Aqueous Solution: Impact on Dissolution Rate. Environ Sci Technol. 2011;45:5260–5266. [PubMed]
9. Kim B, Park C, Murayama M, Hochella MF. Discovery and Characterization of Silver Sulfide Nanoparticles in Final Sewage Sludge Products. Environ Sci Technol. 2010;44:7509–7514. [PubMed]
10. Kaegi R, Voegelin A, Sinnet B, Zuleeg S, Hagendorfer H, Burkhardt M, Siegrist H. Behavior of Metallic Silver Nanoparticles in a Pilot Wastewater Treatment Plant. Environ Sci Technol. 2011;45:3902–3908. [PubMed]
11. Zook JM, Long SE, Cleveland D, Geronimo CLA, MacCuspie RI. Measuring Silver Nanoparticle Dissolution in Complex Biological and Environmental Matrices Using UV-Visible Absorbance. Anal Bioanal Chem. 2011;401:1993–2002. [PubMed]
12. Kent RD, Vikesland PJ. Controlled Evaluation of Silver Nanoparticle Dissolution Using Atomic Force Microscopy. Environ Sci Technol. 2012;46:6977–6984. [PubMed]
13. Lowry GV, Espinasse BP, Badireddy AR, Richardson CJ, Reinsch BC, Bryant LD, Bone AJ, Deonarine A, Chae S, Therezien M, et al. Long-Term Transformation and Fate of Manufactured Ag Nanoparticles in a Simulated Large Scale Freshwater Emergent Wetland. Environ Sci Technol. 2012;46:7027–7036. [PubMed]
14. Li X, Lenhart JJ. Aggregation and Dissolution of Silver Nanoparticles in Natural Surface Water. Environ Sci Technol. 2012;46:5378–5386. [PubMed]
15. Chinnapongse SL, MacCuspie RI, Hackley VA. Persistence of Singly Dispersed Silver Nanoparticles in Natural Freshwaters, Synthetic Seawater, and Simulated Estuarine Waters. Sci Total Environ. 2011;409:2443–2450. [PubMed]
16. Gao J, Powers K, Wang Y, Zhou H, Roberts SM, Moudgil BM, Koopman B, Barber DS. Influence of Suwannee River Humic Acid on Particle Properties and Toxicity of Silver Nanoparticles. Chemosphere. 2012;89:96–101. [PubMed]
17. Li X, Lenhart JJ, Walker HW. Dissolution-Accompanied Aggregation Kinetics of Silver Nanoparticles. Langmuir. 2010;26:16690–16698. [PubMed]
18. Ma R, Levard C, Marinakos SM, Cheng Y, Liu J, Michel FM, Brown GE, Lowry GV. Size-Controlled Dissolution of Organic-Coated Silver Nanoparticles. Environ Sci Technol. 2012;46:752–759. [PubMed]
19. Zhang W, Yao Y, Sullivan N, Chen Y. Modeling the Primary Size Effects of Citrate-Coated Silver Nanoparticles on Their Ion Release Kinetics. Environ Sci Technol. 2011;45:4422–4428. [PubMed]
20. Huynh KA, Chen KL. Aggregation Kinetics of Citrate and Polyvinylpyrrolidone Coated Silver Nanoparticles in Monovalent and Divalent Electrolyte Solutions. Environ Sci Technol. 2011;45:5564–5571. [PMC free article] [PubMed]
21. Ravindran A, Singh A, Raichur AM, Chandrasekaran N, Mukherjee A. Studies on Interaction of Colloidal Ag Nanoparticles with Bovine Serum Albumin (BSA) Colloids surf B, Biointerfaces. 2010;76:32–37. [PubMed]
22. Khan SS, Srivatsan P, Vaishnavi N, Mukherjee A, Chandrasekaran N. Interaction of Silver Nanoparticles (SNPs) with Bacterial Extracellular Proteins (ECPs) and its Adsorption Isotherms and Kinetics. J hazard Mater. 2011;192:299–306. [PubMed]
23. El Badawy AM, Luxton TP, Silva RG, Scheckel KG, Suidan MT, Tolaymat TM. Impact of Environmental Conditions (pH, Ionic Strength, and Electrolyte Type) on the Surface Charge and Aggregation of Silver Nanoparticles Suspensions. Environ Sci Technol. 2010;44:1260–1266. [PubMed]
24. Xiu Z, Ma J, Alvarez PJJ. Differential Effect of Common Ligands and Molecular Oxygen on Antimicrobial Activity of Silver Nanoparticles versus Silver Ions. Environ Sci Technol. 2011;45:9003–9008. [PubMed]
25. Reinsch BC, Levard C, Li Z, Ma R, Wise A, Gregory KB, Brown GE, Lowry GV. Sulfidation of Silver Nanoparticles Decreases Escherichia coli Growth Inhibition. Environ Sci Technol. 2012;46:6992–7000. [PubMed]
26. Kittler S, Greulich C, Diendorf J, Koller M, Epple M. Toxicity of Silver Nanoparticles Increases during Storage Because of Slow Dissolution under Release of Silver Ions. Chem Mater. 2010;22:4548–4554.
27. Xiu Z, Zhang Q, Puppala HL, Colvin VL, Alvarez PJ. Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles. Nano Lett. 2012 doi: 10.1021/nl301934w. [PubMed] [Cross Ref]
28. Levard C, Hotze EM, Lowry GV, Brown GE. Environmental Transformations of Silver Nanoparticles: Impact on Stability and Toxicity. Environ Sci Technol. 2012;46:6900–6914. [PubMed]
29. Wiesner MR, Lowry GV, Casman E, Bertsch PM, Matson CW, Di Giulio RT, Liu J, Hochella MF. Meditations on the Ubiquity and Mutability of Nano-Sized Materials in the Environment. ACS Nano. 2011;5:8466–8470. [PubMed]
30. Bury NR, Wood CM. Mechanism of Branchial Apical Silver Uptake by Rainbow Trout is via the Proton-Coupled Na+ channel. Am J Physiol. 1999;277:R1385–R1391. [PubMed]
31. Solioz M, Odermatt A. Copper and Silver Transport by CopB-ATPase in Membrane Vesicles of Enterococcus hirae. J Biol Chem. 1995;270:9217–9221. [PubMed]
32. Rogers KR, Bradham K, Tolaymat T, Thomas DJ, Hartmann T, Ma L, Williams A. Alterations in Physical State of Silver Nanoparticles Exposed to Synthetic Human Stomach Fluid. Sci Total Environ. 2012;420:334–339. [PubMed]
33. Aaseth J, Olsen A, Halse J, Hovig T. Argyria - Tissue Deposition of Silver as Selenide. Scand J Clin Lab Invest. 1981;41:247–251. [PubMed]
34. Sato S, Sueki H, Nishijima A. Two Unusual Cases of Argyria: the Application of an Improved Tissue Processing Method for X-Ray Microanalysis of Selenium and Sulphur in Ssilver-Laden Franules. Br J Dermatol. 1999;140:158–163. [PubMed]
35. Fisher NM, Marsh E, Lazova R. Scar-Localized Argyria Secondary to Silver Sulfadiazine Cream. J Am Acad Dermatol. 2003;49:730–732. [PubMed]
36. White JML, Powell AM, Brady K, Russell-Jones R. Severe Generalized Argyria Secondary to Ingestion of Colloidal Silver Protein. Clin Exp Dermatol. 2003;28:254–256. [PubMed]
37. Tomi NS, Kranke B, Aberer W. A Silver Man. Lancet. 2004;363:532. [PubMed]
38. Brandt D, Park B, Hoang M, Jacobe HT. Argyria Secondary to Ingestion of Homemade Silver Solution. J Am Acad Dermatol. 2005;53:S105–S107. [PubMed]
39. Trop M, Novak M, Rodl S, Hellbom B, Kroell W, Goessler W. Silver Coated Dressing Acticoat Caused Raised Liver Enzymes and Argyria-Like Symptoms in Burn Patient. J Trauma. 2006;60:648–652. [PubMed]
40. Vlachou E, Chipp E, Shale E, Wilson YT, Papini R, Moiemen NS. The Safety of Nanocrystalline Silver Dressings on Burns: A Study of Systemic Silver Absorption. Burns. 2007;33:979–985. [PubMed]
41. Naqvi AH, Shields JW, Abraham JL. Nasal Argyria (Deposition of Silver-Selenium) in the Photographic Film Industry: Histopathology and Microanalysis. Am J Otolaryngol. 2007;28:430–432. [PubMed]
42. Kim Y, Suh HS, Cha HJ, Kim SH, Jeong KS, Kim DH. A Case of Generalized Argyria after Ingestion of Colloidal Silver Solution. Am J Ind Med. 2009;52:246–250. [PubMed]
43. Chung IS, Lee MY, Shin DH, Jung HR. Three Systemic Argyria Cases after Ingestion of Colloidal Silver Solution. Int J Dermatol. 2010;49:1175–1177. [PubMed]
44. Bowden LP, Royer MC, Hallman JR, Lewin-Smith M, Lupton GP. Rapid Onset of Argyria Induced by a Silver-Containing Dietary supplement. J Cutan Pathol. 2011;38:832–835. [PubMed]
45. Venclíková Z, Benada O, Joska L. Monitoring of Selenium in Oral Cavity Argyria - A Clinical and Microscopic Study. Neuro Endocrinol Lett. 2011;32:286–291. [PubMed]
46. Jonas L, Bloch C, Zimmermann R, Stadie V, Gross GE, Schäd SG. Detection of Silver Sulfide Deposits in the Skin of Patients with Argyria after Long-Term Use of Silver-Containing Drugs. Ultrastruct Pathol. 2007;31:379–384. [PubMed]
47. Loeschner K, Hadrup N, Qvortrup K, Larsen A, Gao X, Vogel U, Mortensen A, Lam HR, Larsen EH. Distribution of Silver in Rats Following 28 Days of Repeated Oral Exposure to Silver Nanoparticles or Silver Acetate. Part Fibre Toxicol. 2011;8:18. [PMC free article] [PubMed]
48. Wijnhoven SWP, Peijnenburg WJGM, Herberts CA, Hagens WI, Oomen AG, Heugens EHW, Roszek B, Bisschops J, Gosens I, Van De Meent D, et al. Nano-Silver – A Review of Available Date and Knowledge Gaps in Human and Environmental Risk Assessment. Nanotoxicol. 2009;3:109–138.
49. Thio BJR, Montes MO, Mahmoud MA, Lee D, Zhou D, Keller AA. Mobility of Capped Silver Nanoparticles under Environmentally Relevant Conditions. Environ Sci Technol. 2012;46:6985–6991. [PubMed]
50. Stebounova LV, Guio E, Grassian VH. Silver Nanoparticles in Simulated Biological Media: A Study of Aggregation, Sedimentation, and Dissolution. J Nanopart Res. 2011;13:233–244.
51. Choi O, Hu Z. Size Dependent and Reactive Oxygen Species Related Nanosilver Toxicity to Nitrifying Bacteria. Environ Sci Technol. 2008;42:4583–4588. [PubMed]
52. Sotiriou GA, Pratsinis SE. Antibacterial Activity of Nanosilver Ions and Particles. Environ Sci Technol. 2010;44:5649–5654. [PubMed]
53. Yang X, Gondikas AP, Marinakos SM, Auffan M, Liu J, Hsu-Kim H, Meyer JN. Mechanism of Silver Nanoparticle Toxicity Is Dependent on Dissolved Silver and Surface Coating in Caenorhabditis elegans. Environ Sci Technol. 2012;46:1119–1127. [PubMed]
54. Marques MRC, Loebenberg R, Almukainzi M. Simulated Biological Fluids with Possible Application in Dissolution Testing. Dissolut Technol. 2011;18:15–28.
55. Trombetta ES, Ebersold M, Garrett W, Pypaert M, Mellman I. Activation of Lysosomal Function During Dendritic Cell Maturation. Science. 2003;299:1400–1403. [PubMed]
56. Schneider LA, Korber A, Grabbe S, Dissemond J. Influence of pH on Wound-Healing: A New Perspective for Wound-Therapy? Arch Dermatol Res. 2007;298:413–420. [PubMed]
57. Thompson WG. Practical Dietetics, with Special Reference to Diet in Disease. 1. Appleton And Company; New York: 1903. Duration of Gastric Digestion of Different Foods; pp. 346–348.
58. Slavin W, Carnrick GR, Manning DC. Chloride Interferences in Graphite-Furnace Atomic-Absorption Spectrometry. Anal Chem. 1984;56:163–168.
59. Espinoza MG, Hinks ML, Mendoza AM, Pullman DP, Peterson KI. Kinetics of Halide-Induced Decomposition and Aggregation of Silver Nanoparticles. J Phys Chem C. 2012;116:8305–8313.
60. Jensen TR, Malinsky MD, Haynes CL, Van Duyne RP. Nanosphere Lithography: Tunable Localized Surface Plasmon Resonance Spectra of Silver Nanoparticles. J Phys Chem B. 2000;104:10549–10556.
61. Song JY, Kim BS. Rapid Biological Synthesis of Silver Nanoparticles Using Plant Leaf Extracts. Bioprocess Biosyst Eng. 2009;32:79–84. [PubMed]
62. Zhao X, Liu R, Teng Y, Liu X. The Interaction between Ag+ and Bovine Serum Albumin: A Spectroscopic Investigation. Sci Total Environ. 2011;409:892–897. [PubMed]
63. Lin S, Chen K, Liang R. Design and Evaluation of Drug-Loaded Wound Dressing Having Thermo Responsive, Adhesive, Absorptive and Easy Peeling Properties. Biomater. 2001;22:2999–3004. [PubMed]
64. Iizaka S, Sanada H, Minematsu T, Oba M, Nakagami G, Koyanagi H, Nagase T, Konya C, Sugama J. Do Nutritional Markers in Wound Fluid Reflect Pressure Ulcer Status? Wound Repair Regen. 2010;18:31–37. [PubMed]
65. Haynes WM, editor. Solubility Product Constants. CRC Handbook of Chemistry and Physics. 91. CRC Press/Taylor and Francis; Boca Raton, FL: 2011. pp. 8–120. (Internet Version)
66. Gustafsson JP. Visual MINTEQ version 3.0: A Chemical Equilibrium Speciation Code; Department of Land and Water Resources Engineering. KTH; Sweden: 2010.
67. Hogstrand C, Wood CM. Toward a Better Understanding of the Bioavailability, Physiology and Toxicity of Silver in Fish: Implications for Water Quality Criteria. Environ Toxicol Chem. 1998;17:547–561.
68. AshaRani PV, Mun GLK, Hande MP, Valiyaveettil S. Cytotoxicity and Genotoxicity of Silver Nanoparticles in Human Cells. ACS Nano. 2009;3:279–290. [PubMed]
69. Poon VKM, Burd A. In vitro Cytotoxity of Silver: Implication for Clinical Wound Care. Burns. 2004;30:140–147. [PubMed]
70. Luoma SN. Silver Nanotechnologies and the Environment: Old Problems or New Challenges? Woodrow Wilson International Center for Scholars; Washington, DC: 2008.
71. Lansdown ABG. A Pharmacological and Toxicological Profile of Silver as an Antimicrobial Agent in Medical Devices. Adv Pharmacol Sci. 2010;2010:910686. [PMC free article] [PubMed]
72. Bal W, Christodoulou J, Sadler PJ, Tucker A. Multi-Metal Binding Site of Serum Albumin. J Inorg Biochem. 1998;70:33–39. [PubMed]
73. Laussac JP, Sarkar B. Characterization of the Copper(II)- and Nickel(II)-Transport Site of Human Serum Albumin. Studies of Copper(II) and Nickel(II) Binding to Peptide 1–24 of Human Serum Albumin by 13C and 1H NMR Spectroscopy. Biochem. 1984;23:2832–2838. [PubMed]
74. Masuoka J, Hegenauer J, Van Dyke BR, Saltman P. Intrinsic Stoichiometric Equilibrium Constants for the Binding of Zinc(II) and Copper(II) to the High Affinity Site of Serum Albumin. J Biol Chem. 1993;268:21533–21537. [PubMed]
75. Bar-Or D, Lau E, Winkler JV. A Novel Assay for Cobalt-Albumin Binding and Its Potential as a Marker for Myocardial Ischemia - A Preliminary Report. J Emerg Med. 2000;19:311–315. [PubMed]
76. Sugio S, Kashima A, Mochizuki S, Noda M, Kobayashi K. Crystal Structure of Human Serum Albumin at 2.5 Å Resolution. Protein Eng. 1999;12:439–446. [PubMed]
77. Shen X, Liang H, Guo J, Song C, He X, Yuan Y. Studies on the Interaction between Ag+ and Human Serum Albumin. J Inorg Biochem. 2003;95:124–130. [PubMed]
78. Adams NWH, Kramer JR. Potentiometric Determination of Silver Thiolate Formation Constants Using a Ag2S Electrode. Aquat. Geochem. 1999;5:1–11.
79. Ballatori N. Glutathione Mercaptides as Transport Forms of Metals. Adv Pharmacol. 1994;27:271–298. [PubMed]
80. Richie JP, Skowronski L, Abraham P, Leutzinger Y. Blood Glutathione Concentrations in a Large-Scale Human Study. Clin Chem. 1996;42:64–70. [PubMed]
81. Odriozola I, Ormategui N, Loinaz I, Pomposo JA, Grande HJ. Coinage Metal-Glutathione Thiolates as a New Class of Supramolecular Hydrogelators. Macromol Symp. 2008;266:96–100.
82. Liu Y, Ma W, Liu W, Li C, Liu Y, Jiang X, Tang Z. Silver(I)-Glutathione Biocoordination Polymer Hydrogel: Effective Antibacterial Activity and Improved Cytocompatibility. J Mater Chem. 2011;21:19214–19218.
83. Cecil R. The Quantitative Reactions of Thiols and Disulphides with Silver Nitrate. Biochem J. 1950;47:572–584. [PubMed]
84. Bellina B, Compagnon I, Bertorelle F, Broyer M, Antoine R, Dugourd P, Gell L, Kulesza A, Mitrić R, Bonačić-Koutecký V. Structural and Optical Properties of Isolated Noble Metal-Glutathione Complexes: Insight into the Chemistry of Liganded Nanoclusters. J Phys Chem C. 2011;115:24549–24554.
85. Bielmyer GK, Bell RA, Klaine SJ. Effects of Ligand-Bound Silver on Ceriodaphnia dubia. Environ Toxicol Chem. 2002;21:2204–2208. [PubMed]
86. Tsipouras N, Rix CJ, Brady PH. Passage of Silver Ions through Membrane-Mimetic Materials, and Its Relevance to Treatment of Burn Wounds with Silver Sulfadiazine Cream. Clin Chem. 1997;43:290–301. [PubMed]
87. Bell RA, Kramer JR. Structural Chemistry and Geochemistry of Silver-Sulfur Compounds: Critical Review. Environ Toxicol Chem. 1999;18:9–22.
88. Gao X, Gao T, Zhang L. Solution-Solid Growth of α-Monoclinic Selenium Nanowires at Room Temperature. J Mater Chem. 2003;13:6–8.
89. Merril CR, Harrington MG. “Ultrasensitive” Silver Stains: Their Use Exemplified in the Study of Normal Human Cerebrospinal Fluid Proteins Separated by Two-Dimensional Electrophoresis. Clin Chem. 1984;30:1938–1942. [PubMed]
90. Furne J, Saeed A, Levitt MD. Whole Tissue Hydrogen Sulfide Concentrations are Orders of Magnitude Lower than Presently Accepted Values. Am J Physiol Regul Integr Comp Physiol. 2008;295:R1479–R1485. [PubMed]
91. Hughes MN, Centelles MN, Moore KP. Making and Working with Hydrogen Sulfide: The Chemistry and Generation of Hydrogen Sulfide in vitro and Its Measurement in vivo: A Review. Free Radic Biol Med. 2009;47:1346–1353. [PubMed]
92. Lee WE, Drago FJ. Toner Treatments for Photographic Images to Enhance Image Stability. J Imag Technol. 1984;10:119–126.
93. Björnstedt M, Odlander B, Kuprin S, Claesson HE, Holmgren A. Selenite Incubated with NADPH and Mammalian Thioredoxin Reductase Yields Selenide, which Inhibits Lipoxygenase and Changes the Electron Spin Resonance Spectrum of the Active Site Iron. Biochem. 1996;35:8511–8516. [PubMed]
94. Klayman DL, Griffin TS. Reaction of Selenium with Sodium-Borohydride in Protic Solvents - Facile Method for Introduction of Selenium into Organic Molecules. J Am Chem Soc. 1973;95:197–199.
95. Beld J, Woycechowsky KJ, Hilvert D. Selenoglutathione: Efficient Oxidative Protein Folding by a Diselenide. Biochem. 2007;46:5382–5390. [PubMed]
96. Haynes WM, editor. Chemical Composition of the Human Body. CRC Handbook of Chemistry and Physics. 91. CRC Press/Taylor and Francis; Boca Raton, FL: 2011. pp. 7–48. (Internet Version)
97. Navarro-Alarcon M, Cabrera-Vique C. Selenium in Food and the Human Body: A Review. Sci Total Environ. 2008;400:115–141. [PubMed]
98. Jeong U, Kim JU, Xia Y. Monodispersed Spherical Colloids of Se@CdSe: Synthesis and Use as Building Blocks in Fabricating Photonic Crystals. Nano Lett. 2005;5:937–942. [PubMed]
99. Johnston HJ, Hutchison G, Christensen FM, Peters S, Hankin S, Stone V. A review of the in vivo and in vitro Toxicity of Silver and Gold Particulates: Particle Attributes and Biological Mechanisms Responsible for the Observed Toxicity. Crit Rev Toxicol. 2010;40:328–346. [PubMed]
100. Lee SM, Lee SH. Generalized Argyria after Habitual Use of AgNO3. J Dermatol. 1994;21:50–53. [PubMed]
101. Westhofen M, Schäfer H. Generalized Argyrosis in Man: Neurotological, Ultrastructural and X-Ray Microanalytical Findings. Arch Otorhinolaryngol. 1986;243:260–264. [PubMed]
102. van der Zande M, Vandebriel RJ, Van Doren E, Kramer E, Rivera ZH, Serrano-Rojero CS, Gremmer ER, Mast J, Peters RJB, Hollman PCH, Hendriksen PJM, Marvin HJP, Peijnenburg AACM, Bouwmeester H. Distribution, Elimination, and Toxicity of Silver Nanoparticles and Silver Ions in Rats after 28-Day Oral Exposure. ACS Nano. 2012;6(8):7427–7442. [PubMed]
103. Glover RD, Miller JM, Hutchison JE. Generation of Metal Nanoparticles from Silver and Copper Objects: Nanoparticle Dynamics on Surfaces and Potential Sources of Nanoparticles in the Environment. ACS Nano. 2011;5:8950–8957. [PubMed]
104. Liu H, Ye Y, Chen J, Lin D, Jiang Z, Liu Z, Sun B, Yang L, Liu J. In situ Photoreduced Silver Nanoparticles on Cysteine: An Insight into the Origin of Chirality. Chem Eur J. 2012;18:8037–8041. [PubMed]
105. Akaighe N, MacCuspie RI, Navarro DA, Aga DS, Banerjee S, Sohn M, Sharma VK. Humic Acid-Induced Silver Nanoparticle Formation Under Environmentally Relevant Conditions. Environ Sci Technol. 2011;45:3895–3901. [PubMed]
106. Hamel SC, Ellickson KM, Lioy PJ. The Estimation of the Bioaccessibility of Heavy Metals in Soils Using Artificial Biofluids by Two Novel Methods: Mass-Balance and Soil Recapture. Sci Total Environ. 1999;244:273–283. [PubMed]
107. Liu X, Sen S, Liu J, Kulaots I, Geohegan D, Kane A, Puretzky AA, Rouleau CM, More KL, Palmore GTR, et al. Antioxidant Deactivation on Graphenic Nanocarbon Surfaces. Small. 2011;7:2775–2785. [PMC free article] [PubMed]